ABSTRACT
The striatin-interacting phosphatase and kinase (STRIPAK) complex is a large, multisubunit protein phosphatase 2A (PP2A) assembly that integrates diverse cellular signals in the Hippo pathway to regulate cell proliferation and survival. The architecture and assembly mechanism of this critical complex are poorly understood. Using cryo-EM, we determine the structure of the human STRIPAK core comprising PP2AA, PP2AC, STRN3, STRIP1, and MOB4 at 3.2-Å resolution. Unlike the canonical trimeric PP2A holoenzyme, STRIPAK contains four copies of STRN3 and one copy of each the PP2AA-C heterodimer, STRIP1, and MOB4. The STRN3 coiled-coil domains form an elongated homotetrameric scaffold that links the complex together. An inositol hexakisphosphate (IP6) is identified as a structural cofactor of STRIP1. Mutations of key residues at subunit interfaces disrupt the integrity of STRIPAK, causing aberrant Hippo pathway activation. Thus, STRIPAK is established as a noncanonical PP2A complex with four copies of regulatory STRN3 for enhanced signal integration.
Subject(s)
Cryoelectron Microscopy , Multienzyme Complexes/metabolism , Multienzyme Complexes/ultrastructure , Protein Multimerization , Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Autoantigens/chemistry , Autoantigens/genetics , Autoantigens/metabolism , Autoantigens/ultrastructure , Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Calmodulin-Binding Proteins/ultrastructure , Hippo Signaling Pathway , Humans , Models, Molecular , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutation , Phosphate-Binding Proteins/chemistry , Phosphate-Binding Proteins/genetics , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/ultrastructure , Phytic Acid/metabolism , Protein Phosphatase 2/chemistry , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/ultrastructure , Protein Serine-Threonine Kinases/chemistry , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Signal TransductionABSTRACT
The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a ß sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the ß sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.
Subject(s)
Inflammasomes/ultrastructure , Multiprotein Complexes/ultrastructure , Phosphate-Binding Proteins/ultrastructure , Pyroptosis/genetics , Animals , Caspase 1/chemistry , Caspase 1/genetics , Caspase 1/ultrastructure , Caspases, Initiator/chemistry , Caspases, Initiator/genetics , Crystallography, X-Ray , HEK293 Cells , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Inflammasomes/genetics , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Phosphate-Binding Proteins/chemistry , Phosphate-Binding Proteins/genetics , Protein Conformation, beta-Strand/genetics , Protein Domains/genetics , Protein Processing, Post-Translational/genetics , ProteolysisABSTRACT
Mycobacterium tuberculosis evades host immune responses by colonizing macrophages. Intraphagosomal M. tuberculosis is exposed to environmental stresses such as reactive oxygen and nitrogen intermediates as well as acid shock and inorganic phosphate (Pi) depletion. Experimental evidence suggests that expression levels of mycobacterial protein PstS3 (Rv0928) are significantly increased when M. tuberculosis bacilli are exposed to Pi starvation. Hence, PstS3 may be important for survival of Mtb in conditions where there is limited supply of Pi. We report here the structure of PstS3 from M. tuberculosis at 2.3-Å resolution. The protein presents a structure typical for ABC phosphate transfer receptors. Comparison with its cognate receptor PstS1 showed a different pattern distribution of surface charges in proximity to the Pi recognition site, suggesting complementary roles of the two proteins in Pi uptake.
